Since the discovered of penicillin, the use of antibiotics to treat the ravages of bacterial infections has saved millions of lives. With the advent of these xe2x80x9cmiracle drugs,xe2x80x9d for a time it was popularly believed that humanity might, once and for all, be saved from the scourge of bacterial infections. In fact, during the 1980s and early 1990s, many large pharmaceutical companies cut back or eliminated antibiotics research and development. They believed that infectious disease caused by bacteria finally had been conquered and that markets for new drugs were limited. Unfortunately, this belief was overly optimistic.
The tide is beginning to turn in favor of the bacteria as reports of drug resistant bacteria become more frequent. The United States Centers for Disease Control announced that one of the most powerful known antibiotics, vancomycin, was unable to treat an infection of the common Staphylococcus aureus (staph). This organism is commonly found in our environment and is responsible for many nosocomial infections. The import of this announcement becomes clear when one considers that vancomycin was used for years to treat infections caused by stubborn strains of bacteria, like staph. In short, the bacteria are becoming resistant to our most powerful antibiotics. If this trend continues, it is conceivable that we will return to a time when what are presently considered minor bacterial infections are fatal diseases.
There are a number of causes for the predicament in which practitioners of medical arts find themselves. Over-prescription and improper prescription habits by some physicians have caused an indiscriminate increase in the availability of antibiotics to the public. The patient is also partly responsible, for even in instances where an antibiotic is the appropriate treatment, patients will often improperly use the drug, the result being yet another population of bacteria that is resistant, in whole or in part, to traditional antibiotics.
The bacterial scourges that have haunted humanity remain, in spite of the development of modern scientific practices to deal with the diseases that they cause. Drug resistant bacteria are now advancing on the health of humanity. A new generation of antibiotics to once again deal with the pending health threat that bacteria present is required.
As more and more bacterial strains become resistant to the panel of available antibiotics, new compounds are required. In the past, practitioners of pharmacology would have to rely upon traditional methods of drug discovery to generate novel, safe and efficacious compounds for the treatment of disease. Traditional drug discovery methods involve blindly testing potential drug candidate-molecules, often selected at random, in the hope that one might prove to be an effective treatment for some disease. The process is painstaking and laborious, with no guarantee of success. Today, the average cost to discover and develop a new drug is nearly US $500 million, and the average time is 15 years from laboratory to patient. Improving this process, even incrementally, would represent a huge advance in the generation of novel antimicrobial agents.
Newly emerging practices in drug discovery utilize a number of biochemical techniques to provide for directed approaches to creating new drugs, rather than discovering them at random. For example, gene sequences and proteins encoded thereby that are required for the proliferation of an organism make for excellent targets since exposure of bacteria to compounds active against these targets would result in the inactivation of the organism. Once a target is identified, biochemical analysis of that target can be used to discover or to design molecules that interact with and alter the functions of the target. Using physical and computational techniques, to analyze structural and biochemical targets in order to derive compounds that interact with a target is called rational drug design and offers great future potential. Thus, emerging drug discovery practices use molecular modeling techniques, combinatorial chemistry approaches, and other means to produce and screen and/or design large numbers of candidate compounds.
Nevertheless, while this approach to drug discovery is clearly the way of the future, problems remain. For example, the initial step of identifying, molecular targets for investigation can be an extremely time consuming task. It may also be difficult to design molecules that interact with the target by using computer modeling techniques. Furthermore, in cases where the function of the target is not known or is poorly understood, it may be difficult to design assays to detect molecules that interact with and alter the functions of the target. To improve the rate of novel drug discovery and development, methods of identifying important molecular targets in pathogenic microorganisms and methods for identifying molecules that interact with and alter the functions of such molecular targets are urgently required.
Escherichia coil represents an excellent model system to understand bacterial biochemistry and physiology. The estimated 4288 genes scattered along the 4.6xc3x97106 base pairs of the Escherichia coli (E. coli) chromosome offer tremendous promise for the understanding of bacterial biochemical processes. In turn, this knowledge will assist in the development of new tools for the diagnosis and treatment of bacteria-caused human disease. The entire E. coli genome has been sequenced, and this body of information holds a tremendous potential for application to the discovery and development of new antibiotic compounds. Yet, in spite of this accomplishment, the general functions or roles of many of these genes are still unknown. For example, the total number of proliferation-required genes contained within the E. coli genome is unknown, but has been variously estimated at around 200 to 700 (Armstrong, K. A. and Fan, D. P. Essential Genes in the metB-malB Region of Escherichia coli K12, 1975, J. Bacteriol. 126: 48-55).
Novel, safe and effective antimicrobial compounds are needed in view of the rapid rise of antibiotic resistant microorganisms. However, prior to this invention, the characterization of even a single bacterial gene was a painstaking process, requiring years of effort. Accordingly, there is an urgent need for more novel methods to identify and characterize bacterial genomic sequences that encode gene products required for proliferation and for methods to identify molecules that interact with and alter the functions of such genes and gene products.
One embodiment of the present invention is a purified or isolated nucleic acid sequence consisting essentially of one of SEQ ID NOs: 1-81, 405-485, wherein said nucleic acid inhibits microorganism proliferation. The nucleic acid sequence may be complementary to at least a portion of a coding sequence of a gene whose expression is required for microorganism proliferation. The nucleic acid sequence may comprise a fragment of one of SEQ ID NOs. 1-81, 405-485, said fragment selected from the group consisting of fragments comprising at least 10, at least 20, at least 25, at least 30, at least 50 or more than 50 consecutive-bases of one of SEQ ID NOs: 1-81, 405-485. The nucleic acid sequence may be complementary to a coding sequence of a gene whose expression is required for microorganism proliferation.
Another embodiment of the present invention is a vector comprising a promoter operably linked to a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs. 1-81, 405-485. The promoter may be active in an organism selected from the group consisting of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae, Helicobacter pylori, Neissera gonorrhoeae, Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus influenzae, Salmonella typhimurium, Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella pneumoniae, Salmonella typhi, Salmonella paratyphi, Salmonella cholerasuis, Staphlyococcus epidermidis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus anthracis, Yersinia pestis, Clostridium botulinum, Campylobacter jejuni, Chlamydia trachomatus, Chlamydia pneumoniae or any species falling within the genera of any of the above species.
Another embodiment of the present invention is a host cell containing the vectors described above.
Another embodiment of the present invention is a purified or isolated nucleic acid consisting essentially of the coding sequence of one of SEQ ID NOs: 82-88, 90-242. One aspect of this embodiment is a fragment of the nucleic acid comprising at least 10, at least 20, at least 25, at least 30, at least 50 or more than 50 consecutive bases of one of SEQ ID NOs: 82-88, 90-242.
Another embodiment of the present invention is a vector comprising a promoter operably linked to the nucleic acids of the preceding embodiment.
Another aspect of the present invention is a purified or isolated nucleic acid comprising a nucleic acid sequence complementary to at least a portion of an intragenic sequence, intergenic sequence, sequences spanning at least a portion of two or more genes, 5xe2x80x2 noncoding region, or 3xe2x80x2 noncoding region within an operon encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 243-357, 359-398.
Another embodiment of the present invention is a purified or isolated nucleic acid comprising a nucleic acid having at least 70% homology to a sequence selected from the group consisting of SEQ ID NOs 1-81, 405-485, 82-88, 90-242 or the sequences complementary thereto as determined using BLASTN version 2.0 with the default parameters. The nucleic acid may be from an organism selected from the group consisting of Staphylococcus aureas, Pseudomonas aeruginosa, Enterobacter cloacae, Helicobacter pylori, Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus influenzae, Salmonella typhimurium, Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella pneumoniae, Salmonella typhi, Salmonella paratyphi, Salmonella cholerasuis, Staphylococcus epidermidis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus anthracis, Yersinia pestis, Clostridium botulinum, Campylobacter jejuni, and Chlamydia trachomatus, Chlamydia pneumoniae or any species falling within the genera of any of the above species.
Another embodiment of the present invention is a purified or isolated nucleic acid consisting essentially of a nucleic acid encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs. 243-357, 359-398.
Another embodiment of the present invention is a vector comprising a promoter operably linked to a nucleic acid encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs.: 243-357, 359-398.
Another embodiment of the present invention is a host cell containing the vector of the preceding embodiment.
Another embodiment of the present invention is purified or isolated polypeptide comprising the sequence of one of SEQ ID NOs: 243-357, 359-398.
Another embodiment of the present invention is purified or isolated polypeptide comprising a fragment of one of the polypeptides of SEQ ID NOs. 243-357. 359-398, said fragment selected from the group consisting of fragments comprising at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 or more than 60 consecutive amino acids of one of the polypeptides of SEQ ID NOs.: 243-357, 359-398.
Another embodiment of the present invention is an antibody capable of specifically binding the polypeptide of the preceding embodiment.
Another embodiment of the present invention is method of producing a polypeptide, comprising introducing a vector comprising a promoter operably linked to a nucleic acid encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs. 243-357, 359-398 into a cell. The method may further comprise the step of isolating said protein.
Another embodiment of the present invention is a method of inhibiting proliferation comprising inhibiting the activity or reducing the amount of a polypeptide having a sequence selected from the group consisting of SEQ ID NOs. 243-357, 359-398 or inhibiting the activity or reducing the amount of a nucleic acid encoding said polypeptide.
Another embodiment of the present invention is method for identifying compounds which influence the activity of a polypeptide required for proliferation comprising:
contacting a polypeptide comprising a sequence selected from the group consisting of 243-357, 359-398 with a candidate compound; and
determining whether said compound influences the activity of said polypeptide.
The activity may be an enzymatic activity. The activity may be a carbon compound catabolism activity. The activity may be a biosynthetic activity. The activity may be a transporter activity. The activity may be a transcriptional activity. The activity may be a DNA replication activity. The activity may be a cell division activity.
Another embodiment of the present invention is a compound identified using the above method.
Another embodiment of the present invention is method for assaying compounds for the ability to reduce the activity or level of a polypeptide required for proliferation, comprising:
providing a target, wherein said target comprises the coding sequence of a sequence selected from the group consisting of SEQ ID NOs. 82-88, 90-242;
contacting said target with a candidate compound; and measuring an activity of said target.
The target may be a messenger RNA molecule transcribed from a coding region of one of SEQ ID. NOs.: 82-88, 90-242 and said activity is translation of said messenger RNA. The target may be a coding region of one of SEQ ID. NOs. 82-88, 90-242 and said activity is transcription of said messenger RNA.
Another embodiment of the present invention is a compound identified using the method above.
Another embodiment of the present invention is a method for identifying compounds which reduce the activity or level of a gene product required for cell proliferation comprising the steps of:
expressing an antisense nucleic acid against a nucleic acid encoding said gene product in a cell to reduce the activity or amount of said gene product in said cell, thereby producing a sensitized cell;
contacting said sensitized cell with a compound, and
determining whether said compound inhibits the growth of said sensitized cell to a greater extent than said compound inhibits the growth of a nonsensitized cell.
The cell may be selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells. The cell may be an E. coli cell. The cell may be from an organism selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae, Helicobacter pylori, Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus influenzae, Salmonella typhimurium, Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella pneumoniae, Salmonella typhi, Salmonella paratyphi, Salmonella cholerasuis, Staphylococcus epidermidis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus anthracis, Yersinia pestis, Clostridium botulinum, Campylobacter jejuni, and Chlamydia trachomatus, Chlamydia pneumoniae or any species falling within the genera of any of the above species. The antisense nucleic acid may be transcribed from an inducible promoter. The method may, further comprise the step of contacting said cell with a concentration of inducer which induces said antisense nucleic acid to a sublethal level. The sub-lethal concentration of said inducer may be such that growth inhibition is 8% or more. The inducer may be isopropyl-1-thio-xcex2-D-galactoside. The growth inhibition may be measured by monitoring optical density of a culture growth solution. The gene product may be a polypeptide. The gene product may be an RNA. The gene product may comprise a polypeptide having a sequence selected from the group consisting of SEQ ID NOs.: 243-357, 359-398.
Another embodiment of the present invention is a compound identified using the method above.
Another embodiment of the present invention is a method for inhibiting cellular proliferation comprising introducing a compound with activity against a gene corresponding to one of SEQ ID NOs.: 82-88, 90-242 or with activity against the product of said gene into a population of cells expressing a gene. The compound may be an antisense oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-81, 405-485, or a proliferation-inhibiting portion thereof. The proliferation inhibiting portion of one of SEQ ID NOs. 1-81, 405-485 may be a fragment comprising at least 10, at least 20, at least 25, at least 30, at least 50 or more than 50 consecutive bases of one of SEQ ID NOs: 1-81, 405-485. The compound may be a triple helix oligonucleotide.
Another embodiment of the present invention is a preparation comprising an effective concentration of an antisense oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-81, 405-485, or a proliferation-inhibiting portion thereof in a pharmaceutically acceptable carrier. The proliferation-inhibiting portion of one of SEQ ID NOs. 1-81, 405-485 may comprise at least 10, at least 20, at least 25, at least 30, at least 50 or more than 50 consecutive bases of one of SEQ ID NOs: 1-81, 405-485.
Another embodiment of the present invention is a method for inhibiting the expression of a gene in an operon required for proliferation comprising contacting a cell in a cell population with an antisense nucleic acid, said cell expressing a gene corresponding to one of SEQ ID NOs.: 82-88, 90-242, wherein said antisense nucleic acid comprises at least a proliferation-inhibiting portion of said operon in an antisense orientation that is effective in inhibiting expression of said gene. The antisense nucleic acid may be complementary to a sequence of a gene comprising one or more of SEQ ID NOs.: 82-88, 90-242. The antisense nucleic acid may be a sequence of one of SEQ ID NOs.: 1-81, 405-485, or a portion thereof. The cell may be contacted with said antisense nucleic acid by introducing a plasmid which expresses said antisense nucleic acid into said cell population. The cell may be contacted with said antisense nucleic acid by introducing a phage which expresses said antisense nucleic acid into said cell population. The cell may be contacted with said antisense nucleic acid by introducing a sequence encoding said antisense nucleic acid into the chromosome of said cell into said cell population. The cell may be contacted with said antisense nucleic acid by introducing a retron which expresses said antisense nucleic acid into said cell population. The cell may be contacted with said antisense nucleic acid by introducing a ribozyme into said cell-population, wherein a binding portion of said ribozyme is complementary to said antisense oligonucleotide. The cell may be contacted with said antisense nucleic acid by introducing a liposome comprising said antisense oligonucleotide into said cell. The cell may be contacted with said antisense nucleic acid by electroporation. The antisense nucleic acid may be a fragment comprising at least 10, at least 20, at least 25, at least 30, at least 50 or more than 50 consecutive bases of one of SEQ ID NOs: 82-88, 90-242. The antisense nucleic acid may be an oligonucleotide.
Another embodiment of the present invention is a method for identifying bacterial strains comprising the steps of:
providing a sample containing a bacterial species; and
identifying a bacterial species using a species specific probe having a sequence selected from the group consisting of SEQ ID NOs. 1-81, 405-485, 82-88, 90-242.
Another embodiment of the present invention is a method for identifying a gene in a microorganism required for proliferation comprising:
(a) identifying an inhibitory nucleic acid which inhibits the activity of a gene or gene product required for proliferation in a first microorganism;
(b) contacting a second microorganism with said inhibitory nucleic acid;
(c) determining whether said inhibitory nucleic acid from said first microorganism inhibits proliferation of said second microorganism; and
(d) identifying the gene in said second microorganism which is inhibited by said inhibitory nucleic acid.
Another embodiment of the present invention is a method for assaying a compound for the ability to inhibit proliferation of a microorganism comprising:
(a) identifying a gene or gene product required for proliferation in a first microorganism;
(b) identifying a homolog of said gene or gene product in a second microorganism;
(c) identifying an inhibitory nucleic acid sequence which inhibits the activity of said homolog in said second microorgansim;
(d) contacting said second microorganism with a proliferation-inhibiting amount of said inhibitory nucleic acid, thus sensitizing said second microorganism;
(e) contacting the sensitized microorganism of step (d) with a compound; and
(f) determining whether said compound inhibits proliferation of said sensitized microorganism to a greater extent than said compound inhibits proliferation of a nonsensitized microorganism.
The step of identifying a gene involved in proliferation in a first microorganism may comprise:
introducing a nucleic acid comprising a random genomic fragment from said first microorganism operably linked to a promoter wherein said random genomic fragment is in the antisense orientation; and
comparing the proliferation of said first microorganism transcribing a first level of said random genomic fragment to the proliferation of said first microorganism transcribing a lower level of said random genomic fragment, wherein a difference in proliferation indicates that said random genomic fragment comprises a gene involved in proliferation.
The step of identifying a homolog of said gene in a second microorganism may comprise identifying a homologous nucleic acid or a nucleic acid encoding a homologous polypeptide in a database using an algorithm selected from the group consisting of BLASTN version 2.0 with the default parameters and FASTA version 3.0t78 algorithm with the default parameters. The step of identifying a homolog of said gene in a second microorganism may comprise identifying a homologous nucleic acid or a nucleic acid encoding a homologous polypeptide by identifying nucleic acids which hybridize to said first gene. The step of identifying a homolog of said gene in a second microorganism may comprise expressing a nucleic acid which inhibits the proliferation of said first microorganism in said second microorganism. The inhibitory nucleic acid may be an antisense nucleic acid. The inhibitory nucleic acid may comprise an antisense nucleic acid to a portion of said homolog. The inhibitory nucleic acid may comprise an antisense nucleic acid to a portion of the operon encoding said homolog. The step of contacting the second microorganism with a proliferation-inhibiting amount of said nucleic acid sequence may comprise directly contacting said second microorganism with said nucleic acid. The step of contacting the second microorganism with a proliferation-inhibiting amount of said nucleic acid sequence may comprise expressing an antisense nucleic acid to said homolog in said second microorganism.
Another embodiment of the present invention is a compound identified using the method above.
Another embodiment of the present invention is a method of assaying a compound for the ability to inhibit proliferation comprising:
(a) identifying an inhibitory nucleic acid sequence which inhibits the activity of a gene or gene product required for proliferation in a first microorgansim;
(b) contacting a second microorganism with a proliferation-inhibiting amount of said inhibitory nucleic acid, thus sensitizing said second microorganism;
(c) contacting the proliferation-inhibited microorganism of step (b) with a compound; and
(d) determining whether said compound inhibits proliferation of said sensitized second microorganism to a greater extent than said compound inhibits proliferation of a nonsensitized second microorganism.
The inhibitory nucleic acid may be an antisense nucleic acid which inhibits the proliferation of said first microorganism. The inhibitory nucleic acid may comprise a portion of an antisense nucleic acid which inhibits the proliferation of said first microorganism. The inhibitory nucleic acid may comprise an antisense molecule against the entire coding region of the gene involved in proliferation of the first microorganism. The inhibitory nucleic acid may comprise an antisense nucleic acid to a portion of the operon encoding the gene involved in proliferation of the first microorganism.
Another embodiment of the present invention is a compound identified using the method above.
Another embodiment of the present invention is a method for assaying compounds for activity against a biological pathway required for proliferation comprising:
sensitizing a cell by expressing an antisense nucleic acid against a nucleic acid encoding a gene product required for proliferation in a cell to reduce the activity or amount of said gene product;
contacting the sensitized cell with a compound; and
determining whether said compound inhibits the growth of said sensitized cell to a greater extent than said compound inhibits the growth of an nonsensitized cell.
The cell may be selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells. The cell may be an E. coli cell. The cell may be an organism selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae, Helicobacter pylori, Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus influenzae, Salmonella typhimurium, Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella pneumoniae, Salmonella typhi, Salmonella paratyphi, Salmonella cholerasuis, Staphylococcus epidermidis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus anthracis, Yersinia pestis, Clostridium botulinum, Campylobacter jejuni, and Chlamydia trachomatus, Chlamydia pneumoniae or any species falling within the genera of any of the above species. The antisense nucleic acid may be transcribed from an inducible promoter. The method may further comprise contacting the cell with an agent which induces expression of said antisense nucleic acid from said inducible promoter, wherein said antisense nucleic acid is expressed at a sublethal level. The sublethal level of said antisense nucleic acid may inhibit proliferation by 8% or more. The agent may be isopropyl-1-thio-xcex2-D-galactoside (IPTG). The inhibition of proliferation may be measured by monitoring the optical density of a liquid culture. The gene product may comprise a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 243-357, 359-398.
Another embodiment of the present invention is a compound identified using the method above.
Another embodiment of the present invention is a method for assaying a compound for the ability to inhibit cellular proliferation comprising:
contacting a cell with an agent which reduces the activity or level of a gene product required for proliferation of said cell;
contacting said cell with said compound; and
determining whether said compound reduces proliferation to a greater extent than said compound reduces proliferation of cells which have not been contacted with said agent.
The agent which reduces the activity or level of a gene product required for proliferation of said cell may comprise an antisense nucleic acid to a gene or operon required for proliferation. The agent which reduces the activity or level of a gene product required for proliferation of said cell may comprise an antibiotic. The cell may contain a temperature sensitive mutation which reduces the activity or level of said gene product required for proliferation of said cell. The antisense nucleic acid may be directed against the same functional domain of said gene product required for proliferation of said cell to which said antisense nucleic acid is directed. The antisense nucleic acid may be directed against a different functional domain of said gene product required for proliferation of said cell than the functional domain to which said antisense nucleic acid is directed.
Another embodiment of the present invention is a compound identified using the method above.
Another embodiment of the present invention is a method for identifying the pathway in which a proliferation-required nucleic acid, or its gene product lies comprising:
expressing a sublethal level of an antisense nucleic acid directed against said proliferation-required nucleic acid in a cell;
contacting said cell with an antibiotic, wherein the a biological pathway on which said antibiotic acts is known; and
determining whether said cell has a substantially greater sensitivity to said antibiotic than a cell which does not express said sublethal level of said antisense nucleic acid.
Another embodiment of the present invention is a method for determining the pathway on which a test compound acts comprising:
(a) expressing a sublethal level of an antisense nucleic acid directed against a proliferation-required nucleic acid in a cell, wherein the biological pathway in which said proliferation-required nucleic acid lies is known,
(b) contacting said cell with said test compound; and
(c) determining whether said cell has a substantially greater sensitivity to said test compound than a cell which does not express said sublethal level of said antisense nucleic acid.
The method may further comprise:
(d) expressing a sublethal level of a second antisense nucleic acid directed against a second proliferation-required nucleic acid in said cell, wherein said second proliferation-required nucleic acid is in a different biological pathway than said proliferation-required nucleic acid in step (a); and
(e) determining whether said cell has a substantially greater sensitivity to said test compound than a cell which does not express said sublethal level of said second antisense nucleic acid.
Another embodiment of the present invention is a purified or isolated nucleic acid consisting essentially of one of SEQ ID NOs: 358, 399-402.
Another embodiment of the present invention is a purified or isolated nucleic acid comprising a sequence selected from the group consisting of 1-81, 405-485, 82-88, 90-242, 358, 399-402.
Another embodiment of the present invention is a compound which interacts with the gene or gene product of a nucleic acid comprising a sequence of one of SEQ ID NOs: 82-88, 90-242 to inhibit proliferation.
Another embodiment of the present invention compound which interacts with a polypeptide comprising one of SEQ ID NOs. 243-357, 359-398 to inhibit proliferation.
Another embodiment of the present invention is a compound which interacts with a nucleic acid comprising one of SEQ ID NOs: 358, 399-402 to inhibit proliferation.